Hi! I have stained with sucess a protein with 27 KDa, but when I tried use the membrane again to stain other protein, I observed the last bands.
How can I remove Ab 1st stained from my membranes? I have ised strip with NaOH 0.1M.
Thanks
0
6 replies to this topic
#2
badcell
-
- Active Members
-
- 94 posts
Enthusiast
1
Neutral
Neutral
Posted 26 January 2005 - 10:31 AM
You can try 62.5 mM Tris pH 6.8, 2% SDS and 100 mM 2-mercaptoethanol. Incubate the membrane at 50ºC for 30 min with ocassional agitation and then wash with TBST and block again.
Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein)
(A.Einstein)
#3
Al Bunsy
-
- Active Members
-
- 6 posts
member
0
Neutral
Neutral
Posted 28 January 2005 - 10:04 AM
5 min H2O
5-10 min 0.2 H NaOH
5 min H2O
Works good.
5-10 min 0.2 H NaOH
5 min H2O
Works good.
#4
Gin
-
- Members
-
- 3 posts
member
0
Neutral
Neutral
Posted 31 March 2005 - 05:30 AM
Hi all,
Usually, after I do the 1st stripping, all most proteins in the membrane disappeared ( I checked it by staining that membrane with Ponceau solution) or just remained a little.
The contents of stripping buffer I used is:
+ 0.2 M Glycine (pH 2.5)
+ 0.05% Tween 20
I incubated the membrane at 80oC for 30 minutes. And then, I washed it 10 min x 3 times with TBST before blocking with skim milk.
What's wrong in my method and how can I solve the problem?
Thank you!
Usually, after I do the 1st stripping, all most proteins in the membrane disappeared ( I checked it by staining that membrane with Ponceau solution) or just remained a little.
The contents of stripping buffer I used is:
+ 0.2 M Glycine (pH 2.5)
+ 0.05% Tween 20
I incubated the membrane at 80oC for 30 minutes. And then, I washed it 10 min x 3 times with TBST before blocking with skim milk.
What's wrong in my method and how can I solve the problem?
Thank you!
Edited by Gin, 31 March 2005 - 05:37 AM.
#5
fred_33
-
- Active Members
-
- 288 posts
Veteran
0
Neutral
Neutral
Posted 31 March 2005 - 06:21 AM
hi
i start to see same problems as you and i use the same protocol of stripping. Hence i'm wondering is "our" glycine too old? mine is more than 3 month...
but for stripping protocol, there was a discussion here
from protein and proteomics forum
fred
i start to see same problems as you and i use the same protocol of stripping. Hence i'm wondering is "our" glycine too old? mine is more than 3 month...
but for stripping protocol, there was a discussion here
from protein and proteomics forum
fred
#6
Gin
-
- Members
-
- 3 posts
member
0
Neutral
Neutral
Posted 31 March 2005 - 06:48 AM
fred_33, on Apr 1 2005, 12:21 AM, said:
hi
i start to see same problems as you and i use the same protocol of stripping. Hence i'm wondering is "our" glycine too old? mine is more than 3 month...
i start to see same problems as you and i use the same protocol of stripping. Hence i'm wondering is "our" glycine too old? mine is more than 3 month...
Thanks for your reply.
Well, but I don't think so. Because another person who taught me that method, he also used the same Glycine with me. And of course, no matter
Thus, I considered the pH of buffer, or the incubate-temperature, or the shaking... are they important to the result of stripping?When you had that problem, how can you overcome it, Fred? You changed with "new" glycine?
Gin,
#7
fred_33
-
- Active Members
-
- 288 posts
Veteran
0
Neutral
Neutral
Posted 31 March 2005 - 07:58 AM
hi
i think ph is not that important caus i use pH 2.7 at 80°C for 30'
but the two last stripping were not by shaking i must admit, and the residual bands apperead just in the pas times.
hence i assume that shaking would increase efficiency
i think ph is not that important caus i use pH 2.7 at 80°C for 30'
but the two last stripping were not by shaking i must admit, and the residual bands apperead just in the pas times.
hence i assume that shaking would increase efficiency
